We have recently cloned 4 CGI PDEs: HFAT and HCAR from human adipose and myocardial CDNA libraries and RcGIP1 and RcGIP2 from rat adipose tissue CDNA libraries. RcGIP1 MRNA is highly expressed in rat and 3T3-L1 adipocytes and its deduced sequence is more closely related to HFAT than to RcGIP2. RcGIP2 MRNA is highly expressed in rat cardiac tissue; its deduced sequence is closely related to that of HCAR. These results are consistent with RcGIP1 (and HFAT) and RcGIP2 (and HCAR) representing two distinct CGI PDE subfamilies, products of two distinct but related genes. To study structure/function relationships of CGI PDEs we have expressed different domains in E. coli as fusion proteins. Catalytic activity for RcGIP1 and HCAR resides in a C terminal region of about 500 amino acids; this region contains the catalytic domain conserved among all PDE families. Deletion of a 44 amino acid segment unique to CGI PDE conserved domains results in a loss of catalytic activity, indicating the importance of this region in CGI PDE activity. Other recombinant fusion proteins will be utilized to define the boundaries of the catalytic domain and putative regulatory domains.